Outcomes of ASFV immunization and challenge in SPF and farm pigs.

(A) Overview of experiment layout marking time points of blood sample collection and performed immunological readouts (dark rectangles indicate collected data). Created with BioRender.com. (B, F) Rectal temperatures and clinical scores were recorded daily after immunization with the attenuated Estonia 2014 strain and after challenge with the highly virulent Armenia 2008 strain. (C, H) Viral DNA levels in blood and serum were measured by qPCR. (D, I) Organs were collected at 17 dpi (D) and at 26 dpc (I) for quantification of viral DNA levels (SM – submandibular lymph node; GH LN – gastrohepatic lymph node). (E) Seroconversion was analyzed by competitive ELISA. For B, C, E, and F, the data points represent values from individual animals and the lines show means of the groups. For D, H, and I, the data points represent values for individual animals. On day 0, the experiment started with nine pigs per group. At 17 dpi, the number was reduced to six per group. By 149 dpi, the number was further decreased to five per group. For B and F, the differences in temperature and clinical scores between two groups were analyzed by comparing areas under curves (AUC) with unpaired t-test. For C, D, and E, the differences between farm and SPF groups were analyzed at each time point by unpaired t-test with Holm-Šídák’s correction for multiple comparisons; *p < 0.05. For G, the survival rates in two groups were compared by Log-rank test. For H and I, the differences in viral loads between protected and non-protected animals were analyzed by unpaired t-test with Holm-Šídák’s correction for multiple comparisons; §p < 0.05; §§§p < 0.001.

Cytokine responses following immunization and challenge, and their correlation with protection.

(A, C) Serum cytokine levels were measured by ELISA. (B, D) Dot plots show correlations between cytokine levels and clinical scores after challenge (r – correlation coefficient; dark red color corresponds to poor clinical outcomes, while gray to protection; correlations with p < 0.1 are outlined with black rectangles). For A, the data points represent values for individual animals and the lines show means of the groups. For C, the data points represent values for individual animals. In A, the differences to baseline measurements (day -5) for farm and SPF pigs were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test (blue hashtags - farm group, pink hashtags - SPF group); #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001. Differences between farm and SPF groups were analyzed at each time point by unpaired t-test with Holm-Šidák’s correction for multiple comparisons; *p < 0.05; **p < 0.01; ****p < 0.0001. In C, the differences to baseline measurements (day -9) for each group were analyzed by mixed-effects analysis with Dunnett’s multiple comparisons test (pink hashtags – SPF group, Ø – non-protected animals); #p < 0.05; Øp < 0.05; ØØp < 0.01; ØØØØp < 0.0001. Differences between farm and SPF groups, as well as protected and non-protected animals, were analyzed at each time point by unpaired t-test with Holm-Šidák’s correction for multiple comparisons.

Cellular immune responses before and after challenge and their correlation with protection.

(A) IFN-γ release from freshly collected PBMCs upon ASFV restimulation was measured by ELISpot before and after challenge (SFC – spot forming cell). Correlations between IFN-γ response and clinical scores after challenge are shown on the plots. (B) T-cell responses upon ASFV in vitro restimulation were analyzed by intracellular cytokine staining. (C) Correlations between the frequencies of cytokine-producing T-cell subsets and clinical scores after challenge are displayed in dot plots (r – correlation coefficient; dark red color corresponds to poor clinical outcomes, while gray to protection; correlations with p < 0.05 are outlined with black rectangles). In A, the data points represent values for individual animals. Measurements were made in triplicate; bars represent mean values for each animal. In B, the data points represent values for individual animals. Differences between farm and SPF groups (*), as well as between protected and non-protected animals (§) were analyzed at each time point by unpaired t-test with Holm-Šidák’s correction for multiple comparisons; *p < 0.05; **p < 0.01; §§p < 0.01.

Blood leukocyte transcriptomic profiles after immunization with the Estonia 2014 strain.

For each group of animals, comparisons were made against the baseline measurements (day 0). Pre-ranked list of DEGs was subjected to GSEA using BTMs as gene sets. (A, B) Dot plots show innate and adaptive BTMs, respectively. Dot size depicts the q-value (FDR), while color represents the normalized enrichment score (NES). FDR < 0.05 was selected as a cutoff.

Correlation of relative BTM expression after Estonia 2014 immunization with protection.

Read counts after bulk RNA-Seq were normalized by the baseline values (day 0). GSVA was then performed to calculate NES values for individual animals using BTMs as gene sets. Obtained NES values were correlated with AUC values of the challenge clinical outcomes. In A and B, the dot plots show innate and adaptive BTMs, respectively. Dot size depicts the p-value, and color represents the correlation coefficient (dark red corresponds to poor clinical outcomes, while gray to protection). Correlations with p < 0.05 are outlined with black rectangles.

Correlation of absolute BTM expression after Estonia 2014 immunization with protection.

GSVA was performed on read counts to calculate NES values for individual animals using BTMs as gene sets. Obtained NES values were correlated with AUC values of the challenge clinical outcomes. In A and B, the dot plots show innate and adaptive BTMs, respectively. Dot size depicts the p-value, and color represents the correlation coefficient (dark red corresponds to poor clinical outcomes, while gray to protection). Correlations with p < 0 .05 are outlined with black rectangles.

Kinetics of average NES of BTM families after Estonia 2014 immunization.

NES values were calculated by GSVA for five farm and five SPF pigs using non-normalized read counts. At each time point, NES values of BTMs belonging to one family were used to determine average family scores. Plots demonstrate average NES values for each family related to either innate (left column) or adaptive immunity (right column). Differences between protected and non-protected farm pigs were analyzed by unpaired t-test with Holm-Šídák’s correction for multiple comparisons; §p < 0.05.

Correlation of absolute BTM expression after Armenia 2008 challenge with protection.

GSVA was performed on read counts to calculate NES values for individual animals using BTMs as gene sets. Obtained NES values were correlated with AUC values of the challenge clinical outcomes. In A and B, the dot plots show innate and adaptive BTMs, respectively. Dot size depicts the p-value, and color represents the correlation coefficient (dark red corresponds to poor clinical outcomes, while gray to protection). Correlations with p < 0 .05 are outlined with black rectangles.

Model of protective immune responses against ASFV challenge in vaccinated pigs.

The findings from BTM expression, systemic cytokine, and T-cell response analyses are integrated. Following immunization with the attenuated virus, an early IFN-α response activates antigen-presenting cells (APCs), triggering sustained cell cycle activation and clonal expansion of antigen-specific lymphocytes. This process generates plasma cells and effector T cells, with efficient memory T helper cell development emerging as a key correlate of protection. Upon challenge, a controlled IFN-α response and a rapid activation of cell cycle genes correlate with protection. The coordinated action of antibody-producing plasma cells and activated helper and cytotoxic T cells effectively controls viral replication while preventing a cytokine storm, ultimately conferring protection against ASFV. Created with BioRender.com.